Composition comprising combination of rapamycin and metformin and use thereof for treating neoplastic diseases

ABSTRACT

A formulation for treating neoplastic diseases, including post-transplant malignancies, genetically-driven neoplasia associated with mTORC1 hyperactivation, and a viral associated malignancy comprising a combination of rapamycin and metformin in a molar ratio of about 20:1, 10:1, 5:1, 4:1, 3:1, or 1:1. A method of treating neoplastic diseases comprising administering the formulation comprising a combination of rapamycin and metformin in a molar ratio of about 20:1, 10:1, 5:1, 4:1, 3:1, or 1:1.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of and claims priority to U.S.application Ser. No. 16/931,133 filed on Jul. 16, 2020 entitled“Composition Comprising Combination Of Rapamycin And An Activator Of AMPKinase And Use Thereof For Treating Disease,” which is a Continuation ofand claims priority to U.S. application Ser. No. 16/040,160, now U.S.Pat. No. 10,952,994, filed on Jul. 19, 2018 entitled “CompositionComprising Combination Of Rapamycin And An Activator Of AMP Kinase AndUse Thereof For Treating Disease,” which is a continuation of U.S.application Ser. No. 15/828,926, now U.S. Pat. No. 10,925,862, filed onDec. 1, 2017 entitled Composition Comprising Combination Of RapamycinAnd An Activator Of AMP Kinase And Use Thereof For Treating Disease,”which is a continuation of U.S. application Ser. No. 15/241,312, nowU.S. Pat. No. 10,765,665, filed on Aug. 19, 2016 entitled “CompositionComprising Combination Of Rapamycin And An Activator Of AMP Kinase AndUse Thereof For Treating Diseases,” which claims the benefit of U.S.Application No. 62/259,384 filed on Nov. 24, 2015 entitled “CompositionComprising Combination Of Rapamycin And An Activator Of AMP Kinase AndUse Thereof For Treating Diseases” and U.S. Application No. 62/318,302filed on Apr. 5, 2016 entitled “Composition Comprising Combination OfRapamycin And An Activator Of AMP Kinase And Use Thereof For TreatingDiseases,” each of which is incorporated herein in its entirety byreference.

FIELD OF THE INVENTION

This invention relates to a composition comprising a combination ofrapamycin and an activator of AMP kinase for use in the treatment and/orprevention of diseases and disease conditions. The invention alsorelates to methods of treating and/or preventing diseases and diseaseconditions.

BACKGROUND

Rapamycin (marketed as sirolimus)) is a macrolide produced by thebacterium Streptomyces hygroscopicus. Sirolimus is currently used as animmunosuppressant and is most often used to prevent rejection oftransplanted organs. Sirolimus has two approved indications—renaltransplantation and lymphangioleiomyomatosis (LAM). Rapamycin has alsobeen shown to be potentially effective in treating Tuberous SclerosisComplex (TSC)-associated seizures, skin disease, brain lesions,pulmonary lesions, and renal lesions. Because rapamycin is animmunosuppressant, long-term (chronic) use of the drug may increase apatient's risk for developing serious side effects (such as infectionand pulmonary toxicity). These findings have raised safety concernsabout the suitability of rapamycin for long-term use.

Rapamycin is an inhibitor of mTOR complex 1 (mTORC1). mTOR (mammaliantarget of rapamycin) is a serine threonine kinase and is a masterregulator of protein synthesis, cell growth, and cell metabolism.Excessive mTORC1 activity has been implicated in multiple diseaseconditions including tuberous sclerosis complex (TSC); polyhydramnios,megalencephaly, and symptomatic epilepsy (PMSE) syndrome; variouscancers including breast, colon, kidney, lung, prostate, pancreatic,etc., inflammatory bowel disease, inflammatory arthritides (multipletypes), inflammatory skin diseases (multiple types), Systemic LupusErythematosis, and neurodegenerative diseases (including Alzheimer'sDisease).

Several studies have indicated that rapamycin may be used to treat orprevent some disease conditions by inhibiting the mTORC1 pathway.Approved rapalogs at current labeling and posology, however, havesignificant safety issues with a risk benefit profile consideredinappropriate for use in patients without an advanced malignancy, organtransplant, or tuberous sclerosis complex.

Thus, there remains a need for safe and effective treatment utilizingrapalogs for these and other non-malignant conditions.

The present drug combination results in increased efficacy withoutincreasing dosage amounts or frequency of doses of rapamycin

SUMMARY

In one aspect, the invention relates to a composition comprising acombination of rapamycin and an activator of AMP kinase for use in thetreatment and/or prevention of diseases and disease conditions. In oneembodiment, the AMP kinase activator is metformin.

In one embodiment the composition is a topical formulation. The topicalformulation may be a gel, an ointment, a cream, or a lotion. In oneembodiment, the topical formulation may include a molar ratio ofrapamycin to metformin in the range of about 20:1 to about 1:1; about20:1 to about 3:1; about 20:1 to about 4:1; about 20:1 to about 5:1;about 15:1 to about 1:1, about 15:1 to about 3:1; about 15:1 to about4:1; about 15:1 to about 5:1; about 10:1 to about 3:1; about 10:1 toabout 4:1; about 10:1 to about 5:1; about 5:1 to about 4:1; about 5:1 toabout 3:1; or about 5:1 to about 1:1.

In another aspect, the invention relates to methods of treating and/orpreventing diseases and disease conditions.

In one embodiment, the method is a method for treating a joint diseasein a subject, wherein the joint disease is characterized byinflammation. The method comprises administering an effective amount ofa topical formulation directly over or on top of an inflamed or diseasedjoint affected by the joint disease. The topical formulation comprises acombination of rapamycin and metformin. In one embodiment the topicalformulation may include a molar ratio of rapamycin to metformin in therange of about 20:1 to about 1:1; about 20:1 to about 3:1; about 20:1 toabout 4:1; about 20:1 to about 5:1; about 15:1 to about 1:1, about 15:1to about 3:1; about 15:1 to about 4:1; about 15:1 to about 5:1; about10:1 to about 3:1; about 10:1 to about 4:1; about 10:1 to about 5:1;about 5:1 to about 4:1; about 5:1 to about 3:1; or about 5:1 to about1:1.

In one embodiment, the topical formulation may be selected from a gel,an ointment, a cream, or a lotion.

In one embodiment, the joint disease is an inflammatory arthritis. Inone embodiment, the inflammatory arthritis is osteoarthritis.

In one embodiment, the method is a method for treating an inflammatoryskin disease in a subject. The method comprises administering aneffective amount of a topical formulation directly to an area of skinaffected by the inflammatory skin disease, the topical compositioncomprising a combination of rapamycin and metformin, wherein the molarratio of rapamycin to metformin is in the range of about 20:1 to about4:1.

In one embodiment, the molar ratio of rapamycin to metformin is in therange of about 20:1 to about 1:1; about 20:1 to about 3:1; about 20:1 toabout 4:1; about 20:1 to about 5:1; about 15:1 to about 1:1, about 15:1to about 3:1; about 15:1 to about 4:1; about 15:1 to about 5:1; about10:1 to about 3:1; about 10:1 to about 4:1; about 10:1 to about 5:1;about 5:1 to about 4:1; about 5:1 to about 3:1; or about 5:1 to about1:1.

In one embodiment, the inflammatory skin disease may be atopicdermatitis (eczema).

In one embodiment, the method is a method of treating an autoimmuneinflammatory skin disease. The autoimmune inflammatory disease may beassociated with systemic lupus erythematosus or discoid lupuserythematosus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting the percent inhibition of ³H-uptake ofdifferent doses of rapamycin and metformin alone or in combination inCaki-1 renal carcinoma cells and shows that substandard doses ofmetformin potentiate rapamycin at 1 nM.

FIG. 2 is a bar graph depicting the percent inhibition of ³H-uptake ofdifferent doses of rapamycin and metformin alone or in combination inCaki-1 renal carcinoma cells and shows that substandard doses ofmetformin potentiate rapamycin at 5 nM.

FIG. 3 is a bar graph depicting the percent inhibition of ³H-uptake ofdifferent doses of rapamycin and methotrexate alone or in combination inCaki-1 renal carcinoma cells and shows that standard doses ofmethotrexate potentiate low doses of rapamycin.

FIG. 4 is a bar graph depicting percent inhibition of ³H-uptake ofdifferent doses of rapamycin and metformin alone in MCF-7 breast cancercells and shows that the effects of rapamycin and metformin alone arenot dose-proportional.

FIG. 5 is a bar graph depicting percent inhibition of 3H-uptake ofdifferent doses of rapamycin and metformin alone or in combination inMCF-7 breast cancer cells and shows that low dose metformin combinedwith low dose rapamycin (1.0 nM) augments inhibition compared torapamycin (1.0 nM) alone.

FIG. 6 is a bar graph depicting percent inhibition of 3H-uptake ofdifferent doses of rapamycin and metformin alone or in combination inMCF-7 breast cancer cells and shows that low dose metformin added to lowdose rapamycin (0.5 nM) augments inhibition compared to rapamycin (0.5nM) alone.

FIG. 7 is a bar graph depicting percent inhibition of 3H-uptake ofdifferent doses of rapamycin and metformin alone or in combination inMCF-7 breast cancer cells and shows that low dose metformin added tovery low dose rapamycin (0.1 nM) adversely affects inhibition comparedto rapamycin (0.1 nM) alone.

FIG. 8 is a graph comparing the percent inhibition of ³H-uptake and themolar ratio of rapamycin to metformin in MCF-7 breast cancer cells. Theoptimal molar ratio range for inhibition of ³H-uptake is between 5:1 and10:1.

DETAILED DESCRIPTION

As used herein, approximating language may be applied to modify anyquantitative representation that may vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term or terms, such as “about” and “substantially,” maynot to be limited to the precise value specified, in some cases.

Rapamycin works by inhibiting the mTORC1 enzyme pathway. This pathway isresponsible for many key cellular processes involved in multiplediseases, including autophagy, HIF-1alpha and VEGF production, cellgrowth, and adaptive immunity. While rapamycin is a frequently used drugfor renal transplant patients, long-term use of rapamycin at standard(typically high) doses can have significant deleterious health effects.

According to one aspect of the invention, the combination of rapamycinwith an enhancing agent augments efficacy and lowers the risk fromunwanted side effects due to excessive rapamycin dosing. Applicant hasdiscovered a complementary agent that acts through an alternativepathway to enhance the efficacy of rapamycin when used alone (i.e., asmonotherapy) for a variety of neoplastic and nonneoplastic diseases,especially when administered topically or locally in a tissue-targeted,site-specific fashion.

By studying the mTORC1 pathway, it was noted that upstream enhancementof an earlier pathway involving the enzyme AMPK (before mTORC1 becomesactive) could enhance the effect of rapamycin within the cell. Multiplecompounds have been found to activate AMPK. For example, a metabolite ofadenosine called AICA riboside indirectly inhibits mTORC1 through aseries of upstream events that involve activation of AMPK. Methotrexate(MTX) increases intracellular concentrations of molecules that activateAMPK such as AICA riboside (5-aminoimidazole-4-carboxamide riboside),AICAR, and ZMP (aminoimidazole carboxamide ribonucleotide). ActivatedAMPK in turn inhibits mTORC1 through at least two discrete pathways.

Metformin, a widely used drug for the first-line treatment of type 2diabetes, indirectly activates AMPK by inhibiting Complex I of themitochondrial respiratory chain. Metformin inhibits ATP synthesis and,thus, increases intracellular levels of ADP and AMP (which leads toactivation of AMPK via LKB1 activation).

Multiple additional compounds have been found to activate AMPK. One ofthese is Permetrexed, an anti-folate drug indicated for non-small celllung cancer and mesothelioma. Permetrexed inhibits the enzymeaminoimidazole caroxamide ribonucleotide formyltransferase (AICART),which in turn leads to the accumulation of AMP, and activation of AMPK.

Without being bound by any specific theory, metformin suppresses mTORC1signaling through both AMPK-dependent and AMPK-independent mechanisms.Because of this upstream series of enzymatic reactions, combining into asingle formulation non-standard (e.g., lower than typically prescribed)doses of rapamycin with an AMPK activator (also at non-standard lowerdoses than is typically prescribed)) may potentiate mTORC1 inhibition.

In one embodiment of the invention, the composition comprises acombination of rapamycin with metformin or another activator of AMPK(for example, methotrexate) to enhance efficacy with respect to mTORC1inhibition, as well as allow for any complementary non-redundantsalutary effects. This combination may be used in the treatment ofrheumatologic, neurodegenerative, and heritable and inborn geneticdiseases, especially when administered topically or locally in atissue-targeted, site-specific fashion.

The popular belief has always been that the mechanism of action (MOA) ofmethotrexate is through inhibition of purine metabolism throughinhibition of dihydrofolate reductase (DHFR). Teachey, et al (Blood(2008)112(5):220-23) was the first to suggest synergy betweenmethotrexate and rapamycin in a non-clinical murine/ex-vivo study ofhuman Acute Lymphoblastic Leukemia (ALL) cells lines. Teachey et al.hypothesized that the MOA of methotrexate is through an upstream effectof inhibiting DHFR activity rather than enhancement of mTOR inhibition.Teachey et al. state that “because resistance to MTX may correlate withhigh DHFR expression, we hypothesized mTOR inhibitors may increasesensitivity of ALL to MTX through decreasing DHFR by increasing turnoverof cyclin D.”

Recognition of the alternative intracellular mechanism of action throughmTOR is crucial in the discovery that methotrexate would be an additiveor synergistic agent with rapamycin and therapeutically important. Acombination drug comprising both rapamycin and methotrexate (ormetformin) in a single dosage form is unknown. It is also unknown thatthe drugs in combination act as a more efficacious mTORC1 inhibitor thaneither drug alone or whether this combination is even safe (i.e., doesnot result in serious dose-limiting toxicity when used long-term). Thiscombination product could be more efficacious than either drug alone andmay result in improvement in safety by allowing for lower doses ofrapamycin to be used chronically, especially when administered topicallyor locally in a tissue-targeted, site-specific fashion.

One aspect of the invention provides a drug combination comprisingrapamycin and any activator of AMP kinase. In one embodiment, the AMPkinase (AMPK) activator may be chosen from compounds such as:methotrexate; metformin; phenformin, a thiazolidinedione; a salicylate;a plant product (e.g., resveratrol from red wine,epigallocatechin-3-gallate from green tea, berberine); AICA riboside;AICAR (5aminoimidazole-4-carboxamide ribonucleotide); or a combinationof two or more thereof. In one embodiment, the AMPK activator ismethotrexate. In another embodiment, the AMPK activator is metformin.

The combination of low (non-standard) dose rapamycin and methotrexatemay be most effective at treating autoimmune and immunologicallymediated inflammatory disorders, while the combination of rapamycin andmetformin may be most effective for patients with diabetes, metabolicsyndrome, and obesity. The combination of rapamycin and metformin mayalso be more effective for Alzheimer's disease and other proteindeposition-associated neurodegenerative diseases, as well as diabeticretinopathy, age-related macular degeneration, osteoarthritis associatedwith type 2 diabetes, chronic liver diseases (including non-alcoholicsteatohepatitis and autoimmune liver diseases, other autoimmunedisorders, chronic non-autoimmune inflammatory disorders, heritable andinborn genetic diseases associated with high mTORC1 activation states,primary and secondary chemoprevention of both malignant andnon-malignant neoplasms, cancer treatment, and chronic pain associatedwith inflammatory diseases. In one embodiment, the chronic pain isassociated with inflammatory arthritis, e.g., osteoarthritis.

Rapamycin is a macrolide produced by the bacterium Streptomyceshygroscopicus having the chemical formula:

In one embodiment, a chemical analog of rapamycin, known as a rapalog,may be used in place of rapamycin. Potentially suitable rapalogs includeeverolimus (Novartis, Afinitor®) and temsirolimus (Wyeth, Torisel®) aswell as still yet to be approved mTORC1 inhibitors (such asridaforolimus (Deforolimus)). As these rapalogs are all derivatives ofrapamycin, it is preferred to use rapamycin.

In one embodiment, a newer second generation rapalog (such as Torin 1,KU0063794, OSE-027) may be used in place of rapamycin. These secondgeneration rapalogs may more potently inhibit mTORC2 as well as mTORC1.

In another embodiment, the agent may inhibit mTORC1, mTORC2, or acombination of both mTORC1 and mTORC2. In yet another embodiment, thecomposition may include an agent that also inhibits other kinases, suchas PI3K, may be used in place of rapamycin (such as NVP-BEZ235,GSK2126458, or PF-04691502) (known collectively as Dual PI3K/mTORinhibitors).

In another embodiment, a rapalog (with or without an AMPK activator suchas metformin) could be combined with an AKT inhibitor (such as AZD5363,GSK690693, Perifosine, and GDC-0068) or a PI3K inhibitor (such asidelalisib, buparlisib, wortmannin, GSK2636771, or GDC-0980[RG7422]).

In one embodiment, the drug combination comprises rapamycin andmetformin. Metformin is a well-known drug commonly used as first-linetreatment of type 2 diabetes mellitus, in addition to being used forpolycystic ovary disease, metabolic syndrome, and diabetes prevention.The chemical name is 1,1-Dimethylbiguanide

The structural formula is:

Use of such a drug combination may substantially reduce the amount ofboth rapamycin and metformin needed for successful treatment of diseasescharacterized by high mTOR activation states, when compared to use ofeither drug alone. Metformin can indirectly inhibit mTORC1 throughdirect activation of AMPK, but also can more directly inactivate mTORC1through inactivating the Ragulator Complex (RagGTPase) and upregulateREDD1 (an mTORC1 inactivator). Metformin activates AMPK, which in turndirectly inactivates mTORC1 (by phosphorylating Raptor), and alsoactivates TSC2 (an mTORC1 inactivator (via Rheb) and tumor suppressorprotein associated with Tuberous Sclerosis Complex).

Metformin also directly enhances autophagy (via phosphorylation of theRaptor-ULK1 complex) as does rapamycin (via phosphorylation of ATG13 andULK1/2). By activating AMPK, use of metformin leads to mTORC1downregulation, IGF-1/AKT pathway downregulation, and p53-mediated cellcycle arrest. These suppressive effects may allow for arrest of tumorprogression or may induce cell death. Metformin also suppresses AKTactivation, suppresses ERK 1/2 signaling, reduces expression of tyrosinekinases (EGFR and HER2), and decreases p-MAPK.

Without being bound to a specific theory, resistance to rapamycin inmalignancies (and other malignant conditions) may result from AKTactivation due to IRS-1 negative feedback on AKT. Metformin inhibits AKTactivation by activating AMPK which in turn phosphorylates and, thus,activates IRS-1 at Ser 789. Combining these two agents (metformin andrapamycin) into a simple formulation (especially at lower than standarddoses of either drug alone) may overcome resistance to rapamycin withoutaggravating clinical safety or incurring dose-limiting toxicity.

Multi-drug chemotherapy regimens (such as doxorubicin, paclitaxel, andcarboplatin) are associated with serious safety issues andtoxicity-precluding widespread use for all but the gravest of malignantconditions. The lower than standard dose combination could augmentchemotherapy (for either treatment or prevention) or could be used as apreferred alternative to standard therapies in appropriately selectedpatients. The drug combination of the present invention may provide asubstantially improved safety profile, allowing for better adherence,compliance, tolerability, and persistence on drug while achievingcomparable efficacy in well-selected patients with appropriate tumortypes and subtypes.

The concept of adding oral standard doses of metformin (500 mg twicedaily) to standard dose (25 mg once weekly) intravenous (parenteral)temsirolimus has been tested in a phase I study (involving a variety ofmalignancies) (MacKenzie M J et al. A phase I study of temsirolimus andmetformin in advanced solid tumors. Invest New Drugs 2012; 30:647-652.)and shown to produce serious and dose-limiting toxicity in all patientsenrolled in the study (N=11), precluding further drug usage at thesedoses in these patients. The drug combination of the present inventioncould substantially improve the safety profile and allow for greatertolerability using relatively low doses of rapamycin with eitherstandard or low doses of metformin (both given orally, locally,topically, or targeted to a specific tissue as a single formulation) fora variety of diseases that include, but are not limited to, both benignand malignant neoplasms.

Everolimus (a rapamycin derivative) was administered together with anoral MEK inhibitor trametinib (GSK1120212) and evaluated in a Phase 1Bstudy in patients with advanced solid tumors (Tolcher A W et al. Annalsof Oncology 2015; 26: 58-64). This trial was based on previous in vitrostudies demonstrating that concomitant use of trametinib and “rapamycin,an mTOR inhibitor, triggered marked cytotoxicity and synergisticeffects”. Concurrent treatment with trametinib and everolimus resultedin frequent treatment-related adverse events, including mucosalinflammation (40%), stomatitis (25%), fatigue (54%), and diarrhea(42%)-side effects frequently seen with everolimus but infrequentlyreported with trametinib. Tolerable combinations of both drugs at dosesadequate to achieve efficacy could not be achieved. Because of thesesubstantial safety issues, the investigators stated that “based on thecurrent data, further investigation of everolimus in combination withtrametinib is not warranted in patients with solid tumors”. Therefore,combining a rapalog with another drug (especially one that affectssignal transduction) neither assures safety nor efficacy and is oftenundesirable.

Cancer stem cells (CSCs) are believed to be a unique and criticalsubpopulation (≤3.5%) of tumor cells capable of self-renewal activity,tumor initiation, and tumor propagation. CSCs are prone to metastasis.Eradicating CSCs is therefore critical to treating (and controlling)cancer. CSCs are resistant to both radiation therapy and chemotherapyand they proliferate despite high doses of such therapies. CSCs are alsoassociated with high mTORC1 activation states and may be targets of thisproposed drug combination.

Tamoxifen is the most widely used hormonal agent for estrogen receptorpositive (ER+) breast cancer. Although ER+ breast cancer cells aresensitive to the therapeutic effects of tamoxifen, breast cancer stemcells, a critically important subpopulation, are resistant to tamoxifenand demonstrates enhanced activation of the mTORC pathway (and itsdownstream targets) when treated with tamoxifen (putting into questionthe ultimate value of tamoxifen hormonal therapy for chemoprevention).mTORC1 activation is associated with many malignancies and theirrespective CSCs, including lung, breast, prostate, colon, and liver. Useof a drug combination comprising low or very low (non-standard) doses ofmetformin and low or very low (non-standard) doses of rapamycin could bea superior therapy for both primary and secondary chemoprevention inpatients at risk for incident and recurrent malignancies. Contrary tostandard dose therapy with rapamycin, the present drug combination atlower than standard doses allows for an optimized safety profile andaddresses both the CSC population of resistant cells as well as thenon-CSC population. Such a lower than standard dose combination isconsistent with a rationally designed, safer therapeutic indicated forlong-term chronic use. Such a combination could be in addition to otherchemopreventative therapies, including hormonal therapies (including ERblockade (e.g., tamoxifen) and aromatase inhibitors (e.g., exemestane)and growth factor receptor antagonists. Such a combination could also beconsidered for use in breast cancer patients who are triple negative(i.e., negative for estrogen receptors, progesterone receptors, and HER2receptors).

In one embodiment, the drug combination comprises rapamycin andmethotrexate. Methotrexate is a known drug and is commonly used to treatvarious cancers, rheumatoid arthritis, psoriasis, and other indications.The chemical name for methotrexate isN-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]L-glutamicacid. The structural formula is:

Use of a drug combination comprising rapamycin and methotrexate mayreduce the amount of both rapamycin and methotrexate needed forsuccessful treatment when compared to use of either drug by itself.Without being bound by a particular theory, the longer the combinationof rapamycin and methotrexate is used, the lower the level of rapamycinneeded in the blood to allow for rapamycin activity (e.g., mTORinhibition). Methotrexate gets polyglutamated within the cell. Up toseven glutamic acid residues can attach to methotrexate. Higher degreesof polyglutamation appear to correlate with enhanced efficacy and areindependent of serum levels of methotrexate. Thus, even when serumlevels of methotrexate are relatively low, the efficacy of themethotrexate remains. Typically efficacy of rapamycin is correlated withblood levels between 5 and 15 ng/mL. Methotrexate polyglutamates gettrapped within the cell and remain active despite low serum levels ofmethotrexate (and low blood levels of rapamycin). Intracellularpolyglutamation of the methotrexate may, thus, improve the efficacy ofrapamycin, even when it is given at lower than standard doses.

This pharmacological effect of methotrexate will potentially allow forlower doses of rapamycin to be given with greater degrees of efficacy ifthe combination is used chronically over weeks to months. This shouldlead to a sustained pharmacodynamic effect due to the maintenance oflong term efficacy in spite of lower dosing of rapamycin which in turnmay result in a safer and more convenient regimen with higher degrees ofoverall efficacy.

The present drug combination could also be used together with the newlydeveloped classes of cancer immunotherapy agents (e.g., checkpointinhibitors) either alone or concomitant with other chemotherapy agentsfor a variety of malignancies.

The present drug combination could also be added onto or combined withother anti-inflammatory agents, such as NSAIDs and PDE4 inhibitors(e.g., apremilast).

Rapamycin is an inhibitor of the mTOR pathway. The mTOR pathway has beenimplicated in a number of diseases and conditions including heritableand inborn genetic conditions; certain malignancies; autoimmunediseases; neurodegenerative diseases; and VEGF-mediated diseases. Thesediseases and conditions may be better treated or prevented using acombination drug that includes both rapamycin and an AMPK activator.

Heritable and inborn genetic diseases that may be associated with themTOR pathway include: autism spectrum disorders; autoimmunelymphoproliferative syndrome; autosomal dominant polycystic kidneydisease; Bannayan-Riley-Ruvalcaba syndrome (BRRS); Birt-Hogg-DubeSyndrome; blue rubber bleb nevus syndrome; COPA syndrome (mutations inthe coatomer subunit alpha (COPA) gene); Cowden disease; epilepsy(Seizure Disorders); cutaneous angiofibromatosis; Duchenne musculardystrophy and other types of muscular dystrophies, myotonic dystrophy,heart failure due to any etiology, diatolic dysfunction, cardiomyopathy,laminopathies, diseases due to mutations in the LMNA gene responsiblefor A-type lamins, dystrophies of skeletal muscle and fat, FamilialAdenomatous Polyposis (FAP); Primary and Secondary Chemoprevention ofmalignancy in FAP; Familial mucocutaneous venous malformations;prevention and treatment of dyskeratosis congenita and its associatedmalignancies (e.g., oropharyngeal carcinoma); prevention and treatmentof Fanconi's Anemia and its associated malignancies (e.g., oropharyngealcancers, myelodysplastic syndrome, and Acute Myeloid Leukemia);prevention and treatment of dyskeratosis; Focal Cortical Dysplasia TypeIIB; Gardner's Syndrome; Turcott's Syndrome; Hutchinson-Gilford progeriasyndrome; Werner syndrome; Familial multiple discoid fibromas; SporadicAngiofibromas; Hypertrophic cardiomyopathy; inborn genetic syndromesassociated with vascular malformations and hemangiomas (including thoseassociated with TIE2 mutations); LEOPARD syndrome (autosomal dominantPTPN11 mutations); Lhermitte-Duclos disease; lymphangioleiomyomatosis(LAM); Lynch Syndrome; lymphatico-vascular malformations; and othercomplicated vascular anomalies; Multiple Endocrine Neoplasia Types I andII; Neurofibromatosis type 1; Neurofibromatosis type II (associated withneural tumors, e.g., schwannomas); Pachyonychia (congenital);Peutz-Jeghers syndrome; Progeria; progeria-like syndromes; ProteusSyndrome; Proteus-like Syndrome; Juvenile polyposis; PTENHamartoma-Tumor Syndromes (PHTS); PMSE (polyhydramnios, megalencephaly,and symptomatic epilepsy) syndrome; Sturge-Weber Syndrome; Seizuresassociated with Sturge-Weber Syndrome; TIE2-mutated venousmalformations; Tuberous Sclerosis Complex (TSC); Cutaneous angiofibromasassociated with TSC; Hypopigmented Lesions associated with TSC;lymphangioleiomyomatosis associated with TSC; Renal Angiomyolipomaassociated with Tuberous Sclerosis Complex; Seizures associated withTSC; Subependymal Giant Cell Astrocytoma (SEGA) Lesions associated withTSC; spontaneous venous malformations. A combination drug that includesrapamycin (or a rapalog) and an AMPK activator may be used to treat orprevent these conditions, or to treat or prevent the symptoms of theseconditions.

Certain malignancies may also be associated the with mTOR pathwayincluding early and Advanced Breast Cancer; bladder cancer anduroepithelial cancers; early and Advanced Renal cell carcinoma; earlyand Advanced Pancreatic cancers and pancreatic neuroendocrine tumors;pancreatic tumors; B cell malignancies and lymphomas; intraocularlymphoma; Childhood astrocytoma; cholangiocarcinomas; colon cancer(including inborn and heritable conditions that increase the risk forcolon cancer); colorectal cancer; colon cancer associated withinflammatory bowel disease; cancer involving any area of thegastrointestinal tract; oropharyngeal cancer; esophageal cancer;Barrett's Esophagus; dermatofibromas; desmoid tumors; desmoid-typetumors; endometrial cancer; epidermal (skin) cancer; Erdheim-Chesterdisease (a rare form of non-Langerhans' cell histiocytosis), facialangiofibromas; angiofibromas; Gliomas; Glioblastoma; Head and NeckCancer; Hemangiomas; Hepatocellular carcinomas; Histiocytosis X;Langherhans Cell Histiocytosis; Letterer-Siwe Disease; HypereosinophilicSyndromes; Leukemias (including acute and chronic leukemias); Lymphoma(of any type); pre-lymphoma conditions (e.g., those associated withSjogren's Syndrome); lung cancer (of any histological type);Lymphangioleiomyomatosis (LAM); mantle cell lymphoma; cutaneous T celllymphoma; Sezary Syndrome and Mycoses fungoides; Parapsoriasis, lichenplanus; melanoma; non-melanoma skin cancer (including basal cellcarcinomas); myeloid neoplasms; Nasopharyngeal Carcinoma; OropharyngealCancer; Osteosarcoma; Port Wine Stains; prostate cancer; Spider Veins;Cherry Angiomas; plasma cell dyscrasias including MGUS (monoclonalgammopathy of undetermined significance), Smoldering Multiple Myeloma,Multiple Myeloma, and Waldenstrom's Macroglobulinemia; RenalAngiomylipoma; renal cell carcinomas and malignancies of anyhistological type; sarcomas; and Midline Granuloma. A combination drugthat includes rapamycin (or a rapalog) and an AMPK activator may be usedto treat or prevent these conditions, or to treat or prevent thesymptoms of these conditions. Chemoprevention of these conditions(including both benign and malignant neoplasms of any pathological orhistological type) can include either primary or secondarychemoprevention or both. Primary prevention may be used to prevent acondition from occurring and treatment may begin before a diagnosis ismade. Secondary prevention may be used in patients who have or had acondition, in which the condition was treated resulting in remission, acure, or the condition being considered “under control.” Secondaryprevention may be used to prevent the development of a second primary orsecond new event or recurrence.

Autoimmune diseases and syndromes may also be associated with the mTORpathway and include immunologically mediated conditions, inflammatoryconditions and autophagy related disorders. These conditions mayinclude: Antiphospholipid antibody syndrome; Autoimmune cytopenias;Autoimmune hemolytic anemia; Autoimmune hepatitis; Autoimmunelymphoproliferative syndrome; Atopic dermatitis; Ankylosing spondylitisand Axial spondyloarthropathies; acne vulgaris, Bronchial asthma;Bronchial Asthma with Eosinophila; Crohns disease; Ulcerative Colitis;Inflammatory Bowel Disease; Celiac Disease; Dermatomyositis;polymyositis; dermatomyositis/polymyositis syndromes associated with anyparticular autoantibody; CREST Syndrome; Type 1 Diabetes Mellitus;Discoid lupus; lupus panniculitis; bullous lupus erythematosus; subacutecutaneous lupus erythematosus; systemic lupus erythematosus (SLE);cutaneous lupus erythematosus; lupus nephritis; cytopenias associatedwith SLE; central nervous system SLE (CNS Lupus); Eosinophilicesophagitis; Eosinophilic Granulomatosis with Polyangiitis (EGPA; ChurgStraus Syndrome); Evan's syndrome; Graft vs. Host Disease;Humoral/Cellular immunodeficiency disorders; hidradenitis supporativa;hypopigmentation disorders involving the skin, Immune thrombocytopenicpurpura (Autoimmune Thrombocytopenic Purpura; IdiopathicThrombocytopenic Purpura); Juvenile idiopathic Arthritis of any type;keloids and scars; non-alcoholic steatohepatitis (NASH); Osteoarthritis;Osteoarthritis Associated With Diabetes Mellitus and Insulin ResistantStates; Psoriasis; Psoriatic Arthritis; Renal Transplantation;Rheumatoid arthritis; SLE (systemic lupus erythematosis); Bullouspemphigus; Pemphigus vulgaris; Bullous pemphigoid; Neuromyelitis optica(NMO); Myasthenia gravis; Multiple sclerosis; Guillain Barre syndrome;CIDP (chronic inflammatory demyelinating polyneuropathy); Opticneuritis; Sarcoidosis; unstable angina pectoris and unstable plaque withatherosclerosis and coronary heart disease; aortic aneurysm; Uveitis ofmultiple etiologies; primary and secondary Sjogrens Syndrome; PrimaryBiliary Cholangitis (Primary Biliary Cirrhosis); Isolated CNSvasculitis; Autoimmune Hepatitis; Peripheral DemyelinatingPolyneuropathy; Behcet's syndrome; Takayasu's arteritis; Giant cellarteritis/temporal arteritis; Mixed cryoglobulinemia; Polymyalgiarhematica; Idiopathic inflammatory myopathies/myositis; Granulomatosisand polyangiitis (Wegener's granulomatosis); Microscopic polyangiitis(MPA); ACPA positive vasculitis; Cogan's syndrome; Autoimmunesensorineural hearing loss; vitiligo; melasma; joint diseasecharacterized by inflammation; and chronic pain associated withinflammatory diseases including inflammatory arthritis. A combinationdrug that includes rapamycin (or a rapalog) and an AMPK activator may beused to treat or prevent these conditions, or to treat or prevent thesymptoms of these conditions. The drug combination may also be used as asteroid sparing agent for any of the above disorders. The drugcombination may also be used to treat post-operative pain caused byinflammation.

In one embodiment of the invention, the drug combination may be used totreat a joint disease characterized by inflammation. Such diseasesinclude inflammatory arthritis of any etiology and are characterized byinflammation of the synovial membrane. Topical application of the drugcombination of rapamycin and metformin may be directly over or on top ofan inflamed or diseased joint to provide the most effective treatment.

The mTOR pathway has also been implication in neurodegenerativediseases, protein deposition diseases; and autophagy related diseases.These diseases and disease conditions include Alzheimer's disease; Betaamyloidopathies; dementia; Head-trauma related dementia; Huntingdondisease; Tauopathies; Parkinson's disease; Presenile dementia; AmyloidAssociated Vasculopathies; Frontotemporal Dementia; Lewy Body Dementia;Amyotrophic Lateral Sclerosis (ALS); and refractory seizures. Acombination drug that includes rapamycin (or a rapalog) and an AMPKactivator may be used to treat or prevent these conditions, or to treator prevent the symptoms of these conditions. The drug combination mayalso be used for neuroprotection, including neuroprotection post-strokeand after head trauma.

Other diseases or conditions associated with the mTOR pathway includevascular endothelial growth factor (VEGF) mediated diseases or diseasesassociated with VEGF and HIF-1 alpha. VEGF is a signal protein producedby cells that stimulates vasculogenesis and angiogenesis. HIF-1 alphastimulates VEGF and is associated with high VEGF states. mTOR activationleads to high levels of HIF-1 alpha production. Laser treatment ofcutaneous vascular lesions results in activation of mTOR andoverproduction of VEGF. Overproduction of VEGF can contribute to diseaseand disease conditions including Age-related macular degeneration;Angiofibromas; blue rubber bleb nevus syndrome; capillary venousmalformations; complex vascular neoplasms; disseminated neonatalhemangiomata; cavernous hemangiomas; Fibrotic conditions; Epithelioidhemangioendothelioma; Diabetic retinopathy; Diabetic nephropathy;diffuse telangiectatic facial erythema; erythrotelangiectatic rosacea;diffuse microcystic lymphatic malformations; fibromas; spider veins;hemangioendotheliomas; Klippel-Trenauney syndrome; hemangioendothelioma;Maffucci Syndrome associated with hemangioendothelioma; Infantilehemagioma; PHACE syndrome associated with infantile hemangioma;cutaneous vascular lesions that tend to recur after treatment withlasers (i.e.; use our combination product to prevent recurrence of thesevascular lesions (e.g., port wine stains) following use of lasers,especially pulsed dye lasers); Complications from Intra-arterial stents(for use in stents to prevent thrombosis); Kaposi's sarcoma (KS) (bothskin and systemic manifestations) (KS associated with organtransplantation, HIV, or seen in elderly people); kaposiformhemangioendothelioma; port wine stains; port wine stains associated withSturge-Weber Syndrome; spider veins; cherry angiomata; skintransplantation; telangiectases; vascular malformations occurring denovo or associated with an inborn genetic mutation (e.g., TIE2 mutation;rosacea. A combination drug that includes rapamycin (or a rapalog) andan AMPK activator may be used to treat or prevent these conditions, orto treat or prevent the symptoms of these conditions. The drugcombination may also be used as a VEGF sparing agent for any of theabove disorders.

Inhibition of the mTOR pathway also has been associated with increasedlongevity and delaying of aging in laboratory animals. The use of anAMPK activator such as metformin or methotrexate in combination withrapamycin may allow for a lower dose of rapamycin to be used as aneffective prophylactic antiaging therapy. Such prophylaxis could resultin either extension of life or a healthier aging process with fewerassociated co-morbidities such diabetes, cardiovascular disease, heartfailure and diastolic dysfunction, and dementia. Such a drugcombination, especially when administered topically, could be used forskin rejuvenation and treatment/prevention of wrinkling and reduction ofthe effects of sun damage and UV light exposure.

In an aspect of the invention, the combination drug may be used to treatany condition associated with the mTOR pathway, including heritable andinborn genetic diseases, malignancies, renal transplantation, autoimmuneand immunologically mediated inflammatory diseases, neurodegenerativediseases, and VEGF mediated diseases.

In one embodiment, the combination drug may be used to treat tuberoussclerosis complex (TSC). TSC is a rare condition that affects25,000-40,000 patients in the United States and up to 2 million patientsglobally. It occurs in 1:6000 births and is caused by a genetic mutationin either of 2 oncogene suppressors leading to excessive mTOR complex 1(mTORC1) activity. Symptoms of the condition vary but can includesystemic hamartomas (large and small tumorous growths) on the brain,eye, skin, heart, lungs, and kidneys and intractable seizures from CNSlesions leading to cognitive decline. There is currently no approvedtherapeutic agent for modifying the course of the disease includingseizures, cognitive decline, and behavioral disorders. Cutaneous TSCdisease is a progressive disease that results in a visible manifestationof the multi-organ disease and can result in significant and disfiguringskin conditions.

In another embodiment, the combination drug may be used to treatpolyhydramnios, megaencephaly, and symptomatic epilepsy (PMSE) syndrome.PMSE syndrome is an ultra-rare neurodevelopmental disorder found amongthe Old Order Mennonite population. It is caused by a homozygousdeletion of exons 9 to 13 of the LYK5/STRADA gene responsible forencoding a protein called STRADα (pseudokinase STE20-related kinaseadaptor a), an upstream inhibitor of mTORC1. PMSE syndrome ischaracterized by infantile-onset, treatment-resistant multifocalepilepsy, severe cognitive delays, and craniofacial dysmorphism.Patients with PMSE syndrome face an increased risk of death fromepilepsy and 38% of PMSE patients die before age 6. There are currentlyno approved therapeutic agents for altering the course of the condition.

In one embodiment, the combination drug may be used to treat early stageAlzheimer's Disease (AD). Rapamycin has been shown to induce autophagythrough mTOR inhibition and significantly reduce plaques, tangles, andcognitive defects in mice when given before the establishment of plaquesand tangles in the brain. Majumder, et al., PLoS ONE, 2011; 9(6):e25416.Thus, the combination drug may be more effective at preventing thedevelopment of beta-amyloid plaques when used early in the diseaseprocess.

In one embodiment, the combination drug may be used to treat Kaposi'ssarcoma (KS). KS is a human herpesvirus 8 associated malignancyassociated with immunosuppression. It is most commonly seen in patientswith HIV/AIDS, and transplant patients. There are geographic and ethnicpredispositions to KS.

In one embodiment, the inflammatory skin disease may be atopicdermatitis (eczema).

In one embodiment, the combination drug may be used to treat two relatedautoimmune diseases: skin disease associated with systemic lupuserythematosus (SLE) and discoid lupus erythematosus (DLE). Theseconditions are autoimmune inflammatory diseases that affect the skin andproduce persistent scaly, disk-like plaques on the scalp, face, and earsand may cause pigmentary changes, scarring, and hair loss.

In one embodiment, the combination drug may be used to treatpachyonychia congenita, an ultra-rare autosomal dominant disease thataffects several thousand people worldwide. The disease is caused by amutation in one of five keratin genes: K6a, K6b, K6c, K16, or K17. Thedisease causes focal palmoplantar hyperkeratosis, hypertrophic naildystrophy, follicular hyperkeratosis, and oral leukokeratosis. It doesnot affect lifespan. Trials have been conducted to assess whetherrapamycin might be effective in treating pachyonychia congenital. Thesystemic side effects of oral rapamycin limited the trials. Use of thecombination drug would reduce these systemic side effects by reducingthe amount of rapamycin in each dose, by reducing the frequency ofdosing, or both.

In one embodiment, the combination drug may be used to treat a PTENHamartoma Tumor Syndrome, which includes Cowden Syndrome (CS)Lhemitte-Duclos disease (LD), Bannayan-Riley-Ruvalcaba syndrome (BRRS),osteoarthritis, and Proteus Syndrome (PS).

Any route of administration of the combination drug may be effective intreating TSC. For example an oral or parenteral (subcutaneous,intramuscular, or intravenous) route of administration may be effectivein treating the progression of the disease and to control or preventsymptoms such as seizures and cognitive decline. A topical treatment maybe used to treat cutaneous TSC. Proper formulation and dosages willdepend on the specifics of the patient (age, weight, etc.).

Proper formulation and dosages will depend on the disease condition tobe treated. The drug combination may be administered in any manner thatis suitable for treating the desired condition.

Any suitable dosage formulation for administering the combination may beused for treatment. For example, the combination may be incorporatedinto a tablet, capsule, or a liquid formulation for oral administration;a cream, lotion, ointment, gel, or paste for topical administration; orin a liquid, lyophilized form, or nanoparticle form for parenteraladministration; or a viscous base for intraarticular administration(e.g., together with hyaluronic acid).

The above dosage forms will also include the necessary carriermaterial(s), excipient(s), lubricant(s), buffer(s), or the like.

The drug combination of the present invention may be used inpharmaceutical compositions. These pharmaceutical compositions can beused in the preparation of individual, single unit dosage forms.Pharmaceutical compositions and dosage forms can comprise the drugcombination as described herein or a pharmaceutically acceptable salt orsolvate thereof. Pharmaceutical compositions and dosage forms canfurther comprise one or more carriers, excipients or diluents.

Non-limiting examples of possible dosage forms include tablets; caplets;capsules, such as soft elastic gelatin capsules; cachets; troches;lozenges; dispersions; suppositories; powders; aerosols (e.g., nasalsprays or inhalers); topical dosage forms such as gels; creams, lotions,and ointments; liquid dosage forms suitable for oral or mucosaladministration to a patient, including suspensions (e.g., aqueous ornon-aqueous liquid suspensions, oil-in-water emulsions or a water-in-oilliquid emulsions), solutions and elixirs.

The pharmaceutical compositions comprising the drug combinations may besuitable for oral administration and can be presented as discrete dosageforms, such as, but not limited to, tablets (e.g., chewable tablets),caplets, capsules and liquids (e.g., flavored syrups). Such dosage formscontain predetermined amounts of active ingredients as described hereinand can be prepared by methods of pharmacy well known to those skilledin the art.

Such dosage forms can be prepared by any of the methods of pharmacy. Ingeneral, pharmaceutical compositions and dosage forms for oraladministration are prepared by uniformly and admixing the activeingredients with liquid carriers, finely divided solid carriers or bothand then shaping the product into the desired presentation if necessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Typical oral dosage forms are prepared by combining the activeingredients in an admixture with at least one excipient according toconventional pharmaceutical compounding techniques. Excipients can takea wide variety of forms depending on the form of preparation desired foradministration. Non-limiting examples of excipients suitable for use inoral liquid or aerosol dosage forms include water, glycols, oils,alcohols, flavoring agents, preservatives and coloring agents.Non-limiting examples of excipients suitable for use in solid oraldosage forms (e.g., powders, tablets, capsules and caplets) includestarches, sugars, micro-crystalline cellulose, diluents, granulatingagents, lubricants, binders and disintegrating agents.

Non-limiting examples of excipients that can be used in oral dosageforms include binders, fillers, disintegrants and lubricants.Non-limiting examples of binders suitable for use in pharmaceuticalcompositions and dosage forms include corn starch, potato starch orother starches, gelatin, natural and synthetic gums such as acacia,sodium alginate, alginic acid, other alginates, powdered tragacanth,guar gum, cellulose and its derivatives (e.g., ethyl cellulose,cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethylcellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinizedstarch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),microcrystalline cellulose and mixtures thereof.

Non-limiting examples of suitable forms of microcrystalline celluloseinclude, but are not limited to, the materials sold as AVICEL®(microcrystalline cellulose) PH-101, AVICEL® (microcrystallinecellulose) PH-103, AVICEL RC-581@ (crystalline cellulose andcarboxymethylcellulose sodium), AVICEL® (microcrystalline cellulose)PH-105 (available from FMC Corporation, American Viscose Division,Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. A specific binderis a mixture of microcrystalline cellulose and sodium carboxymethylcellulose sold as AVICEL RC-581@ (crystalline cellulose andcarboxymethylcellulose sodium). Suitable anhydrous or low moistureexcipients or additives include AVICEL-PH-103™® (microcrystallinecellulose) PH-103 and Starch 1500® LM (pregelatinized starch).

Non-limiting examples of fillers suitable for use in the pharmaceuticalcompositions and dosage forms disclosed herein include talc, calciumcarbonate (e.g., granules or powder), microcrystalline cellulose,powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,starch, pregelatinized starch and mixtures thereof. The binder or fillerin pharmaceutical compositions is typically present in from about 50 toabout 99 wt. % of the pharmaceutical composition or dosage form.

Disintegrants are used in the compositions to provide tablets thatdisintegrate when exposed to an aqueous environment. The amount ofdisintegrant used varies based upon the type of formulation and isreadily discernible to those of ordinary skill in the art. Typicalpharmaceutical compositions comprise from about 0.5 to about 15 wt. % ofdisintegrant, from about 1 to about 10 wt. %, or from about 1 to about 5wt. % of disintegrant.

Non-limiting examples of disintegrants that can be used inpharmaceutical compositions and dosage forms include agar-agar, alginicacid, calcium carbonate, microcrystalline cellulose, croscarmellosesodium, crospovidone, polacrilin potassium, sodium starch glycolate,potato or tapioca starch, other starches, pre-gelatinized starch, otherstarches, clays, other algins, other celluloses, gums and mixturesthereof.

Non-limiting examples of lubricants that can be used in pharmaceuticalcompositions and dosage forms include calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil), zincstearate, ethyl oleate, ethyl laureate, agar and mixtures thereof. Whenpresent, lubricants may be used in an amount of less than about 1 wt. %of the pharmaceutical compositions or dosage forms.

The dose administered must be carefully adjusted according to age,weight and condition of the patient, as well as the route ofadministration, dosage form and regimen and the desired result.

In one embodiment, the drug combination may be formulated for oraladministration or sublingual administration. In one embodiment, the drugcombination may comprise rapamycin and methotrexate in a ratio ofapproximately 1:2, approximately 1:2.5, approximately 1:3, approximately1:3.5, or approximately 1:4. In one embodiment, rapamycin may be presentin an amount of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, or even about 9mg. Methotrexate may be present in an amount of about 6 mg, about 9 mg,about 12 mg, about 15 mg, about 18 mg, about 21 mg, about 24 mg, or evenabout 27 mg. Methotrexate may also be given in about 2.5 mg multiples upto about 25 mg (about 2.5 mg, about 5.0 mg, about 7.5 mg, about 10 mg,about 12.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg,and about 25 mg).

In one embodiment rapamycin may be present in an amount ranging fromabout 0.5 mg to about 10 mg and methotrexate in the range of from about3 mg to about 30 mg in the same oral dosage form. In another embodimentrapamycin may be present in an amount ranging from about 0.5 mg to about8 mg and methotrexate in the range of from about 5 mg to about 25 mg inthe same oral dosage form. Here as elsewhere in the specification andclaims, ranges can be combined to form new and non-disclosed ranges.

In one embodiment, the rapamycin may be present in an amount of about0.5 mg, about 1 mg, about 2.0 mg, about 2.5 mg, about 3.0 mg, about 3.5mg, about 4.0 mg, about 4.5 mg, or about 5.0 mg. Methotrexate may bepresent in an amount of about 7.5 mg, about 8.0 mg, about 8.5 mg, about9.0 mg, about 9.5 mg, or about 10.0 mg.

In another embodiment, the drug combination may be used to treatheritable and inborn genetic diseases. A lower or higher dose ofmethotrexate as compared to rapamycin may be suitable to treat orprevent these diseases. The ratio of the combination of rapamycin andmethotrexate may be about 2:1 (rapamycin to methotrexate) or about 1:2;about 1:2.5; about 1:3, about 1:3.5; and about 1:4.

The drug combination may be administered orally up to one tablet perday. Because of the potential side effects of the drugs, especially themethotrexate, it is preferable to administer the drug combination lessfrequently. In one embodiment, the drug combination is administered in aonce weekly dose. In another embodiment, the drug combination isadministered weekly but in two half doses, with the second dose beinggiven 24 hours after the first dose.

The drug combination may be administered orally up to one tablet perday. Sublingual administration may achieve a higher blood level of thedrug combination with a lower dose of drug due to enhanced absorption.Thus, it may be preferred to administer the sublingual formulation onceweekly.

Use of an additive or synergistic agent (e.g., an AMPK activator such asmethotrexate)) with rapamycin may enhance efficacy, lower the requireddose of the rapamycin and/or decrease the frequency of drugadministration. In one embodiment, the drug combination may beadministered in a once weekly pulsed regimen. This dosing regimen mayalleviate potential side effects seen with use of rapamycin,methotrexate, or both including undesired immunosuppression, stomatitis,hyperlipidemia, hyperglycemia, and cytopenia.

In one embodiment, the drug combination may be formulated as a rectalsuppository, especially for treatment of infants and children. The samegeneral dosing regimen as for oral administration would apply to arectal suppository formulation.

In one embodiment, the drug combination may be formulated forintra-articular administration. This formulation may be used to treatjoint diseases characterized by inflammation. In one embodiment, thejoint disease is osteoarthritis (e.g., osteoarthritis of the knee joint)or inflammatory arthritis involving a specific joint. The drugcombination may include viscosupplementation as a base (e.g., hyaluronicacid). The hyaluronic acid may serve as the medium into which the drugcombination is added. In one embodiment, the drug combination ofrapamycin and methotrexate is added to a medium comprising hyaluronicacid. The rapamycin may be added in an amount of about 0.5 mg, about 1.0mg, about 1.5 mg, about 2.0 mg, about 2.5 mg, even about 5.0 mg. Themethotrexate maybe a added in an amount of about 4.0 mg, about 4.5 mg,about 5.0 mg, about 5.5 mg, about 6.0 mg, even about 8.0 mg. Thisformulation may be administered via intra-articular administration intoa joint (e.g., a knee joint) every 2-3 months up to 3 times per year. Inanother embodiment, the drug combination may include rapamycin andmetformin or any other AMPK activator. In another embodiment, the drugcombination may contain rapamycin, methotrexate, and another AMPKactivator.

Tablets of various sizes can be prepared, e.g., of about 2 mg to 2000 mgin total weight, containing both of the active substances in the rangesdescribed above, with the remainder being a physiologically acceptablecarrier of other materials according to accepted pharmaceuticalpractice. These tablets can, of course, be scored to provide forfractional doses. Gelatin capsules can be similarly formulated. Uniquecolor coatings and markings may be used for easy identification forpatients to enhance safety and compliance and create branding.

In one embodiment, the drug combination may be in a liquid formulation.Liquid formulations may be prepared by dissolving or suspending thecombination of active substances in a conventional liquid vehicleacceptable for pharmaceutical administration so as to provide thedesired dosage in one to two teaspoonfuls. Such dosage forms can beadministered to the patient on a regimen of one to two doses per day.Special coloring and flavoring may be added to enhance compliance andcreate branding.

In one embodiment, the drug combination may be formulated for parenteraladministration. As with the oral formulation, the drug combination maycomprise rapamycin and methotrexate in a ratio of approximately 1:2,approximately 1:2.5, approximately 1:3, approximately 1:3.5, orapproximately 1:4 and approximately 1:4.5. In one embodiment, rapamycinmay be present in an amount of about 3 mg, about 4 mg, about 5 mg, about6 mg, about 7 mg, about 8 mg, or even about 9 mg. Methotrexate may bepresent in an amount of about 9 mg, about 12 mg, about 15 mg, about 18mg, about 21 mg, about 24 mg, or even about 27 mg or about 7.5 mg, about10 mg, about 12.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about22.5 mg, about 25 mg, and about 27.5 mg.

In one embodiment, rapamycin may be present in an amount ranging fromabout 0.5 mg to about 10 mg and methotrexate in the range of from about2.5 mg to about 30 mg in the same oral dosage form. In anotherembodiment, rapamycin may be present in an amount ranging from about 0.5mg to about 8 mg and methotrexate in the range of from about 5 mg toabout 25 mg in the same oral dosage form.

In one embodiment, the rapamycin may be present in an amount of about0.5 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3.0mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, or about 5.0 mg.Methotrexate may be present in an amount of about 5.0 mg, about 7.5 mg,about 8.0 mg, about 9.0 mg, or about 10.0 mg.

In another embodiment, the drug combination may be used to treatheritable or inborn genetic diseases. A lower or higher dose ofmethotrexate as compared to rapamycin may be suitable to treat orprevent these diseases. The ratio of the combination of rapamycin andmethotrexate may be about 2:1 (rapamycin to methotrexate) or about 1:2,or about 1:3, or about 1:4, or about 1:5.

In one embodiment, the parenteral (subcutaneous) formulation of the drugcombination may be administered once weekly by injection, including byautoinjection. In another embodiment, the parenteral formulation may beadministered weekly in two half doses, with the second half dose beingadministered 24 hours after administration of the first half dose.

In formulating the compositions, the active substances, in the amountsdescribed above, are compounded according to accepted pharmaceuticalpractice with a physiologically acceptable vehicle, carrier, excipient,binder, preservative, stabilizer, flavor, etc., in the particular typeof unit dosage form.

Illustrative of the excipients which may be incorporated in tablets arethe following: a binder such as gum tragacanth, acacia, corn starch orgelatin; an excipient such as dicalcium phosphate or cellulose; adisintegrating agent such as corn starch, potato starch, alginic acid orthe like; a lubricant such as stearic acid or magnesium stearate; asweetening agent such as sucrose, aspartame, lactose or saccharin; aflavoring agent such as orange, peppermint, oil of wintergreen orcherry. When the dosage unit form is a capsule, it may contain inaddition to materials of the above type a liquid carrier such as a fattyoil. Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets orcapsules may be coated with shellac, sugar or both. A syrup of elixirmay contain the active compound, water, alcohol or the like as thecarrier, glycerol as solubilizer, sucrose as sweetening agent, methyland propyl parabens as preservatives, a dye and a flavoring such ascherry or orange.

Sustained release forms of such formulations may be used and may providesuch amounts biweekly, weekly, monthly, etc. A dosing period of at leastone to two weeks is required to achieve minimal benefit.

In one embodiment, the drug combination may be formulated for topical(cutaneous), localized, tissue-targeted, or site-specificadministration. Such formulations may be in the form an ointment, cream,lotion, gel, or paste. Topical formulations also include eye drops andnasal sprays. Such dosages may include rapamycin in the amount of about0.01%, about 0.025%, about 0.05%, about 0.75%, about 0.1%, about 0.25%,about 0.5%, about 0.75%, about 1.0%, about 1.5%, about 2.0%, about 2.5%,about 3.0%, about 3.5%, or about 4.0% by weight of rapamycin. In oneembodiment, the amount of rapamycin is in the range of about 0.01% toabout 4.0% by weight; about 0.1% to about 3.5% by weight; about 0.25 toabout 3.0% by weight; about 0.5% to about 2.5% by weight; about 0.75% toabout 2.0% by weight; about 1.0% to about 2.5% by weight. Metformin (ormethotrexate) may be present in this dosage in an amount of about 0.1%,about 0.2%, about 0.5%, about 1.0%, about 1.5%, about 2.0%, or about2.5% by weight of metformin (or methotrexate).

In one embodiment, a formulation of the drug combination may include amolar ratio of rapamycin to metformin in the range of about 20:1 toabout 1:1; about 20:1 to about 3:1; about 20:1 to about 4:1; about 20:1to about 5:1; about 15:1 to about 1:1, about 15:1 to about 3:1; about15:1 to about 4:1; about 15:1 to about 5:1; about 10:1 to about 3:1;about 10:1 to about 4:1; about 10:1 to about 5:1; about 5:1 to about4:1; about 5:1 to about 3:1; or about 5:1 to about 1:1. In oneembodiment, the formulation is a topical formulation.

In one embodiment, a formulation of the drug combination may includerapamycin in an amount of up to 4.0 grams. In another embodiment, theformulation may include rapamycin in a range of about 0.01 to about 4.0grams; about 0.1 grams to about 3.5 grams; about 0.25 to about 3.0grams; about 0.5 to about 2.5 grams; about 0.7 to about 2.0 grams; orabout 1.0 to about 2.5 grams. In one embodiment, the formulation of thedrug also may include metformin in an amount of up to 0.7 grams. Inanother embodiment, the formulation of the drug also may includemetformin in a range of about 0.7 grams to about 0.00007 grams, about0.7 grams to about 0.0007 grams, about 0.7 grams to about 0.007 grams,about 0.7 grams to about 0.45 grams, about 0.7 grams to about 0.028grams, about 0.6 grams to about 0.02 grams, or about 0.6 grams to about0.01 grams. In one embodiment, the formulation is a topical formulation.

In one embodiment, the topical formulation is a gel comprising rapamycinand an activator of AMP kinase such as methotrexate or metformin. In oneembodiment, the gel comprises a combination of rapamycin and metforminas the active ingredient and at least one excipient.

In one embodiment, the molar ratio of rapamycin to metformin in the gelformulation is in the range of about 20:1 to about 1:1; about 20:1 toabout 3:1, about 20:1 to about 4:1; about 20:1 to about 5:1; about 15:1to about 1:1, about 15:1 to about 3:1; about 15:1 to about 4:1; about15:1 to about 5:1, about 10:1 to about 3:1; about 10:1 to about 4:1;about 10:1 to about 5:1; about 5:1 to about 4:1; about 5:1 to about 3:1;or about 5:1 to about 1:1. In one embodiment, the gel formulationcontains rapamycin in the range of about 0.1 grams to about 4.0 gramsand metformin in the range of about 0.00007 grams to about 0.60 grams.In another embodiment, the gel formulation contains rapamycin in therange of about 1.0 grams to about 3.0 grams and metformin in the rangeof about 0.007 grams to about 0.42 grams.

The gel formulation also includes excipients. Suitable excipientsinclude emulsifiers, organogelators and emollients. Emulsifiers includepolyethylene glycol stearate, a glycol stearate, a glyceryl stearate,cetearyl alcohol and ceteareth 20, methylcellulose, Cetomacrogol 1000,and lecithin. Suitable organogelators include 4-tertbutyl-1-arylcyclohecanols derivatives, polymeric (e.g. poly(ethylene glycol),polycarbonate, polyesters, and poly(alkylene), Gemini gelators (e.g.N-lauroyl-L-lysine ethyl ester), Boc-Ala(1)-Aib(2)-.beta.-Ala(3)-OMe(synthetic tripeptide), and low molecular weight gelators (e.g. fattyacids and n-alkanes). Suitable emollients include cetostearyl alcohol,cetyl alcohol, isopropyl palmitate, caprylic/capric triglyceride, PPG-2myristyl ether propionate, dimethicone, methicone, petrolatum, lanolin,and mineral oil.

If desired, other additives including surfactants, penetrationenhancers, preservatives, viscosity modifiers, and emulsion stabilizersmay be included in the mannitol compositions. Suitable surfactantsinclude sodium lauryl sulfate, cetostearyl alcohol, ceteareth 12,ceteareth 20, cetearyl alcohol, Cetomacrogol 1000, stearic acid, andpoloxamer. Suitable penetration enhancers include propylene glycol.Suitable preservatives include methylparaben, propylparaben,ethylhexylglycerin, phenoxyethanol, chlorocresol, potassium sorbate,sorbic acid, bronopol, methychloroisothiazolinone, andmethylisothiazolinone. Suitable viscosity modifiers includecarboxymethylcellulose, carboxyethylcellulose, acrylate crosspolymer,and carbomer. Suitable emulsion stabilizers include xanthan gum,glyceryl stearate, and carbomer.

Other additives may be added such as aromatic agents, antiseptics, andcolorants

In one embodiment, a topical formulation of the drug combination mayinclude rapamycin and at least two AMPK activators. In one embodiment, atopical formulation of the drug combination includes rapamycin,methotrexate, and about 0.5% to about 27.0% salicylic acid in 0.5%increments by weight of salicylic acid.

In one embodiment, a topical formulation may include topical rapamycinand topical metformin. Rapamycin would be used in a concentrationranging from about 0.1% to about 2.5% and metformin would be used in aconcentration ranging from about 0.02% to about 1.5%. These ranges wouldallow usage of these drugs topically for many different indications.

The drug combination may also be administered through intravitreal, orsubconjunctival injection, or topically. For intravitreal orsubconjunctival injection, the methotrexate dose would be about 200 mcgper 0.1 mL or about 400 mcg per 0.1 mL. For intravitreal orsubconjunctival injection, the rapamycin dose would be about 50 pg permL to about 200 mcg/mL. Intravitreal injections of the combination couldalso include rapamycin at a dose of about 350 mcg to about 450 mcg everytwo months as needed or subconjunctival injections of rapamycin at adose of about 1300 mcg at days 0, 60 and 120. For topical ophthalmicadministration, the dose of rapamycin would be about 50 pg per mL toabout 50 mcg per mL and the methotrexate dose would be about 200 mcg per0.1 mL in a formulation which may be applied at bedtime and throughoutthe day. The topical formulation may be in a solution, a suspension, anemulsion, and it may be administered in the form of eyedrops, a cream,an ointment, a gel, or an injectable to the eye and/or the eyelid. Thecombination containing rapamycin may also be administered in milligramquantities as a surgical implant that could be contained in a diffusiblewalled reservoir embedded and sutured to the wall of the sclera orcontained within a carrier system such as liposomes to allow for slowrelease of the drug combination.

In one embodiment, the drug combination may comprise rapamycin andmetformin. This combination may be preferable for patients withdiabetes, metabolic syndrome, hyperlipidemia, and/or methotrexateintolerance, as well as for chemoprevention of both malignant andnon-malignant neoplastic and non-neoplastic diseases, neurodegenerativediseases such as Alzheimer's disease, and multiple autoimmune andchronic inflammatory disorders. Metformin would be given in a dosage ina range of about 100 mg to about 1000 mg or about 300 mg to about 900mg. The metformin may be present in amount of about 150 mg, about 300mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900mg, or about 1000 mg. The metformin may be present in an amount of about166 mg, about 333 mg, about 666 mg, about 1200 mg or about 200 mg, about400 mg, about 800 mg, or about 1200 mg. Rapamycin would be given in adosage of about 0.15 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about1.1 mg, about 1.2 mg, about 1.3, mg, about 1.4 mg, 1.5 mg, about 2.0 mg,about 2.5 mg, and about 3.0 mg. This formulation could be given orallyas a once daily pill. Other dosing regimens may be suitable fordifferent indications.

In one aspect of the invention, a patient in need of treatment may beevaluated with a companion diagnostic to determine whether treatmentwith a combination drug would be efficacious. The method includesdetermining a patient's baseline mTOR activation state (including theactivation state of mTORC1 downstream pathway components). A finding ofa high mTOR activation state would indicate a greater likelihood ofsuccess. A finding of normal or low mTOR activity state would indicate alower likelihood for success. After several weeks of treatment, anychange in the patient's mTOR activation state will help determine if thegiven dose is the correct dose. If mTOR activity has not dropped, anincreased dose may be necessary. If drug resistance is suspected,treatment may be stopped. This pharmacodynamics biomarker of responsewill help guide therapy. Tissue-specific mTOR activation state may beassumed using a biomarker (e.g., an imaging biomarker such as MRI orultrasound to determine the presence or absence of synovitis),immunohistochemistry (using biopsy tissue), or a localized, salivary,secretory, or peripheral blood biomarker yet to be determined.

The change in mTOR activation state from baseline (e.g., at weeks 2, 4,or 6) may be used to predict future outcome (success) at a later pointin time (e.g., at 24-48-52 weeks). This is a predictive biomarker ofresponse.

The phosphorylation status of downstream activation proteins that getphosphorylated by mTORC1 (such as p70S6 ribosomal kinase; S6 protein;4E-BPI, and eIF4B) and mTOR itself (phospho-mTOR) may be evaluated (byeither immunohistochemistry of biopsied tissue or FACS (fluorescentactivating cell sorting) analysis of circulating lymphocytes or otherformed elements) to determine high mTOR activation states. Rapamycinwould inhibit mTORC1 and the phosphorylation of downstream proteins butno change would occur in the total level of these proteins.

mTORC1 inhibition promotes autophagy. Inadequate autophagy is associatedwith many of the diseases and conditions being targeted by the proposeddrug combinations. A companion diagnostic for these drug combinationswould determine baseline levels/states of autophagy and the effect onsuch levels posttreatment. The determination of autophagy level/stateusing specific biomarkers of autophagy would allow for better patientprofiling of who would and would not benefit from the drug combinationboth at baseline and at early treatment. Prediction of long termresponse is a potential outcome of such an autophagy biomarker as wellas its use to guide adequate dosing.

Examples

³H-Thymidine Uptake Assay

Renal Cell Carcinoma

Pilot plating experiments using Human kidney clear cell carcinoma cellline Caki-I (HTB-46). The cells were exposed to various dosages ofrapamycin, methotrexate, and metformin (see Tables below for dosages) toassess inhibition of DNA synthesis/replication. 0.1% DMSO, the testarticle solvent, in media was the control.

The cells were passaged onto 24-well plates at a concentration of 30,000cells/well, in total medium volume of 1 mL (RPMI 1640+10%FBS+antibiotics). The cells were then allowed to attach and wereincubated from 24 hours before treatment. Cells were then treated, intriplicate, with various concentrations of treatment articles.

³H-thymidine (1 mCi/mL) was diluted to 1 μCi in 5 μL of medium (RPMI1640+10%/FBS+antibiotics) and then added to each well at either 18- or42-hr post treatment, for the 24- and 48-hr time points respectively.

Cells were then washed three times with 1 ML of DPBS at 24- or 48-hrpost treatment to remove unincorporated ³H-thymidine. Each well of cellswas then lysed in 0.5 mL of 0.5N NaOH+0.5% SDS. Lysate was thentransferred to scintillation tubes containing 1 mL if scintillationfluid for counting (3 min/sample).

The results of the pilot plating experiments are summarized below.

TABLE 1 Pilot Plating Experiment-Human kidney clear cell carcinoma cellline Caki-I 24-hr post treatment 48-hr post treatment Treatment ArticleAverage cpm Average cpm Rapamycin 20 nm 12044 6349 Rapamycin 10 nm 126117463 Rapamycin 5 nm 14805 8584 Rapamycin 0 nm 27457 12818 Methotrexate20 nm 26144 11273 Methotrexate 10 nm 27266 20251 Methotrexate 5 nm 3114816772 Methotrexate 2.5 nm 30659 4811 Methotrexate 0 nm 27457 12818Metformin 50 nm 30699 11141 Metformin 20 nm 28060 10624 Metformin 10 nm34530 13671 Metformin 5 nm 29246 12001 Metformin 1 nm 29394 10074Metformin 0 nm 65587 13295

It was noted that the 48-hr incorporation was less than the 24-hrincorporation, suggesting that the cells had been growth arrested or hadovergrown the well and sloughed off.

For the next phase of the experiment, the Caki-1 cells were tested withthe following treatment articles and evaluated at 24 hours posttreatment:

-   -   Rapamycin: 10 nM, 5 nM, 1 nM    -   Methotrexate: 100 nm    -   Metformin: 5 nm, nm, 0.5 nm    -   Rapamycin+methotrexate: 5 nm+100 nm, 1 nm+100 nm    -   Rapamycin+metformin: 5 nM+5 nM, 5 nM+1 nM, 5 nM+0.5 nM, 1 nM+5        nM, 1 nM+1 nM, 1 nM+0.5 nM    -   Control: 0.1% DMSO+media

Results of the treatments are summarized in Table 2 and FIGS. 1-3 .

TABLE 2 ³H-Uptake in Human kidney clear cell carcinoma cell line Caki-ITreatment Average Article Dosage Cpm 1 Cpm 2 Cpm 3 Cpm Rapamycin  10 nM9861 8987 8486 9111 Rapamycin  5 nM 8771 7473 10030 8758 Rapamycin  1 nM10730 9692 11531 10651 Metformin  5 nM 17424 13187 7921 15306 Metformin 1 nM 18473 14784 15710 16322 Metformin  0.5 nM 19450 16142 16208 17267Methotrexate 100 nM 10636 12192 4932 11414 Control  Media + DMSO 1833313675 8579 16004 Rapamycin + 5 nM + 5 nM 6348 7191 6985 6841 metforminRapamycin + 5 nM + 1 nM 9023 6397 7401 7607 metformin Rapamycin +   5nM + 0.5 nM 8549 7403 4229 7976 metformin Rapamycin + 1 nM + 5 nM 1257113180 5759 12876 metformin Rapamycin +  1M + 1 nM 3661 7519 10361 8940metformin Rapamycin +   1 nM + 0.5 nM 7749 10699 9373 9274 metforminRapamycin +   5 nM + 100 nM 7784 6636 8506 7642 methotrexate Rapamycin + 1 nM + 100 nM 8471 5828 3434 7150 methotrexate

Cells treated with a combination of either rapamycin+metformin orrapamycin+methotrexate resulted in lower thymidine uptake compared toindividual treatments with the notable exception of rapamycin+metforminat 1 nM+5 nM where the incorporation was slightly higher than rapamycinalone.

The results demonstrate a synergistic effect for combination treatmentswith the combination of rapamycin and metformin. Additivity was observedfor the combination of rapamycin and methotrexate.

The results of Phase 2 showed that metformin alone had no effect on theinhibition of 3H-thymindine uptake at 5 nM, 1 nM, or 0.5 nM. When usedin combination with rapamycin, however, metformin appeared to enhancethe inhibitory response of rapamycin by approximately 30% relative torapamycin alone. This was unexpected and is indicative of synergy.

Breast Cancer Human Cell Lines

Human breast adenocarcinoma cell line (MCF-7 (HTB-22) cells were exposedto various dosages of rapamycin, methotrexate, and metformin, eitheralone or in combination (see Tables below for dosages) to assessinhibition of DNA synthesis/replication. 0.1% DMSO, the test articlesolvent, in media was the control.

Possible augmentation of the inhibitory effects of rapamycin at verylow, but clinically relevant doses was assessed by adding metformin tolower than standard doses of rapamycin to test its effect on inhibitionof ³H-thymidine uptake.

The cells were passaged onto 24-well plates at a concentration of 50,000cells/well, in total medium volume of 1 mL (DMEM+10% FBS+0.01 mg/mLinsulin+antibiotics). The cells were then allowed to attach and wereincubated from 24 hours before treatment. Cells were then treated, intriplicate, with various concentrations of treatment articles.

³H-thymidine (1 mCi/mL) was diluted to 1 μCi in 5 μL of medium (DMEM+10%FBS+0.01 mg/mL insulin+antibiotics) and then added to each well at 18-hrpost treatment.

Cells were then washed three times with 1 ML of DPBS at 24- or 48-hrpost treatment to remove unincorporated ³H-thymidine. Each well of cellswas then lysed in 0.5 mL of 0.5N NaOH+0.5% SDS. Lysate was thentransferred to scintillation tubes containing 1 mL if scintillationfluid for counting (3 min/sample).

The cells were tested with the following treatment articles andevaluated at 24 hours post treatment:

-   -   Rapamycin: nM, 0.5 nM, 0.1 nM    -   Metformin: 0.5 nM, 0.1 nM, 0.05 nM    -   Rapamycin+metformin: 0.1 nM+0.5 nM, 0.5 nM+0.5 nM, 0.1 nM+0.5        nM, 1 nM+0.1 nM, 0.5 nM+0.1 nM, 0.1 nM+0.1 nM, 1 nM+0.05 nM, 0.5        nM+0.05 nM, 0.1 nM+0.05 nM

Table 3 and FIGS. 4-7 summarize the results.

TABLE 3 ³H-Uptake in Human breast adenocarcinoma cell line(MCF-7)Treatment Average Article Dosage Cpm 1 Cpm 2 Cpm 3 Cpm Rapamycin   1 nM3114 1220 2548 2294 Rapamycin  0.5 nM 2537 2524 2567 2543 Rapamycin  0.1nM 1041 1516 986 1181 Metformin  0.5 nM 1283 1048 921 1084 Metformin 0.1 nM 2238 2595 1444 2092 Metformin 0.05 nM 1267 1123 1041 1144Control Media 3722 3432 3013 3389 Rapamycin +  1 nM + 0.5 nM 1374 15901495 1486 metformin Rapamycin + 0.5 nM + 0.5 nM  1568 1676 2320 1855metformin Rapamycin + 0.1 nM + 0.5 nM  3435 3917 3925 3759 metforminRapamycin +  1 nM + 0.1 nM 1149 862 1641 1217 metformin Rapamycin + 0.5nM + 0.1 nM  1547 1089 963 1200 metformin Rapamycin + 0.1 nM + 0.1 nM 2403 1351 1762 1839 metformin Rapamycin +   1 nM + 0.05 nM 1371 13691311 1350 metformin Rapamycin + 0.5 nM + 0.05 nM 937 1291 909 1046metformin Rapamycin + 0.1 nM + 0.05 nM 1463 1771 1071 1435 metformin

The standard therapeutic serum trough levels of rapamycin considered tobe clinically relevant for current uses is 5-20 ng/mL. The standard dosetherapeutic serum Cmax for metformin is much higher at 1-2 microgram/mL.As can be seen in the above Table 3, the dosages used in theseexperiments for both rapamycin and metformin fall well below theselevels.

At these lower doses of both rapamycin and metformin, the effects ofrapamycin and metformin alone are not dose proportional.

Low dose metformin augments the inhibitory effect of rapamycin (1.0 nM)vs rapamycin alone. The lease effective does of metformin (0.1 nM) morethan doubles the inhibitory effect of rapamycin at 1.0 nM (64% vs. 32%)and rapamycin at 0.5 nM (65% vs. 25%). (See FIGS. 5 and 6 .)

FIGS. 4-7 depict bar graphs representing the percent inhibition of³H-uptake for each concentration. The data indicate that when the ratioof rapamycin to metformin is too low (e.g., less than 5:1), thecombination loses efficacy. (See FIGS. 7 and 8 ). Thus, the percentinhibition of ³H-thymidine uptake (e.g., DNA synthesis) is ratiodependent, not dose-dependent. (Table 4.)

TABLE 4 Comparison of Efficacy of Rapamycin to Metformin Ratios Rationof 1:5 1:1 2:1 5:1 10:1 20:1 Rapamycin to Metformin Rapa (nM)/ 0.1/0.50.1/0.1 0.1/0.05 0.5/0.1 0.5/0.05 1.0/0.5 Metformin (nM) % −11%* 46% 58%65% 69% 60% Inhibition Rapa (nM)/ 1.0/0.5 1.0/0.1 Metformin (nM) %Inhibition 56% 64% *An in verted ratio of 1:5 was antagonistic.

While the invention has been described with reference to variousexemplary embodiments, it will be appreciated that modifications mayoccur to those skilled in the art, and the present application isintended to cover such modifications and inventions as fall within thespirit of the invention.

The invention claimed is:
 1. A method of treating a neoplastic diseaseor disorder comprising administering an effective amount of acomposition comprising a combination of rapamycin and metformin to asubject, wherein the molar ratio of rapamycin to metformin is in therange of about 20:1 to about 1:1.
 2. The method of claim 1, wherein themolar ratio of rapamycin to metformin is in the range of about 15:1 toabout 3:1.
 3. The method of claim 1, wherein the molar ratio ofrapamycin to metformin is in the range of about 10:1 to about 4:1. 4.The method of claim 1, wherein the molar ratio of rapamycin to metforminis about 5:1 to about 3:1.
 5. The method of claim 1, wherein the molarratio of rapamycin to metformin is about 5:1 to about 4:1.
 6. The methodof claim 1, wherein the neoplastic disease or disorder is selected froma post-transplant malignancy, a genetically-driven neoplasia associatedwith mTORC1 hyperactivation, and a viral associated malignancy.
 7. Themethod of claim 6, wherein the neoplastic disease or disorder is apost-transplant malignancy selected from the group consisting of skincancer, Kaposi's sarcoma, hepatocellular carcinoma, and renal cellcarcinoma.
 8. The method of claim 6, wherein the neoplastic disease ordisorder is a genetically-driven neoplasia associated with mTORC1hyperactivation selected from the group consisting of familialadenomatous polyposis (FAP), colon cancer, vascular neoplasms andanomalies, PTEN Hamartoma Tumor Syndrome (PHTS), PIK3CA mutations,mTORC1 gain-of function mutations, hypereosinophilic syndrome, renalcell carcinoma, Peutz-Jeghers Syndrome, Erdheim-Chester Disease, ChronicMyelogenous Leukemia, tuberous sclerosis complex (TSC),lymphangioleiornyomatosis (LAM), and Neuroendocrine tumors (NETs). 9.The method of claim 6, wherein the neoplastic disease or disorder is aviral associated malignancy selected from the group consisting of humanherpes virus-8 (HHV-8) associated malignancies, hepatocellularcarcinoma, lymphoproliferative syndromes, and breast cancer.
 10. Themethod of claim 1, wherein the composition is formulated for oraladministration.
 11. The method of claim 1, wherein the composition isformulated for topical administration.
 12. The method of claim 1,wherein the composition is formulated for parenteral administration. 13.The method of claim 11, wherein the composition comprises a nanoparticleencapsulating at least the rapamycin, the metformin, or the combinationof rapamycin and metformin.
 14. A method of treating a neoplasticdisease or disorder mediated by mTORC1 activation comprisingadministering an effective amount of a composition comprising acombination of rapamycin and metformin to a subject, wherein the molarratio of rapamycin to metformin is in the range of about 20:1 to about1:1.
 15. The method of claim 14, wherein the molar ratio of rapamycin tometformin is in the range of about 10:1 to about 4:1.
 16. The method ofclaim 15, wherein the composition comprises a nanoparticle encapsulatingat least the rapamycin, the metformin, or the combination of rapamycinand metformin.
 17. The method of claim 14, wherein the neoplasticdisease or disorder is selected from a post-transplant malignancy, agenetically-driven neoplasia associated with mTORC1 hyperactivation, anda viral associated malignancy.
 18. The method of claim 17, wherein theneoplastic disease or disorder tuberous sclerosis complex (TSC).
 19. Themethod of claim 17, wherein the neoplastic disease or disorder islymphangioleiomyomatosis (LAM) occurring either sporadically or as partof TSC.
 20. The method of claim 14, wherein the neoplastic disease ordisorder is a gastrointestinal neoplasm.